Shigella is a major cause of diarrhea, dysentery, and mortality annually worldwide. S. flexneri is a CDC/NIAID Category B priority pathogen. It causes disease by invading and spreading through the colonic mucosa. The bacteria enter cells by inducing a process that resembles macropinocytosis. Once within the cell, they move to the cell periphery by means of actin-based motility. At the cell periphery, S. flexneri pushes out against the plasma membrane, forming a protrusion that contains the bacterium at its tip. The protrusion is then engulfed by an adjacent cell, whereupon the cycle of actin-based motility and cell-to-cell spread is repeated. Whereas the mechanisms by which S. flexneri enters cells have been studied extensively, the molecular mechanisms involved in S. flexneri spread from one cell into an adjacent cell are poorly understood. In this application, we propose a detailed investigation of S. flexneri intercellular spread that utilizes both targeted and genomewide approaches. Our targeted approaches will test the hypothesis that the cellular pathway of filopodia extension, which requires diaphanous formins and VASP, is used by S. flexneri to enhance its intercellular spread. Our genome-wide approach will examine the human genome for additional factors required for intercellular spread. Our specific aims are: 1. Characterize the role of the S. flexneri effector OspE2 and eukaryotic VASP in intercellular spread; 2. Characterize the role of diaphanous formins in the formation of plasma membrane protrusions by S. flexneri; and, 3. Identify and characterize other host factors required for S. flexneri intercellular spread using a genome-wide human siRNA screen; We believe that these approaches will lead to insights not only into the mechanisms of intercellular spread of S. flexneri, but also into fundamental mechanisms of eukaryotic cellular and intercellular processes.